The present invention is generally in the area of assays involving detection and/or measurement of endothelial cell protein C/activated protein C receptor or soluble forms thereof derived either by proteolysis or by alternative splicing.
The activation of protein C to its active serine protease, activated protein C (APC), initiates a series of events that play a key role in the regulation of blood coagulation. The clinical importance of the protein C pathway is evidenced by the multitude of dysfunctions in this pathway that result in thrombosis (Esmon and Schwarz. 1995. Trends Cardiovasc. Med. 5:141-148; Reitsma, et al. 1995. Thromb. Haemost. 73:876-879). Patients deficient in protein C usually exhibit life threatening thrombotic-complications in infancy (Seligsohn et al., 1984. N. Engl. J. Med. 310, 559-562; Esmon, 1992. Trends Cardiovasc. Med. 2, 214-220) that are corrected by protein C administration (Dreyfus et al., 1991. N. Engl. J. Med. 325, 1565-1568).
Protein C and APC have also been implicated in the regulation of the host response to inflammation. Activated protein C (APC) can prevent the lethal effects of E. coli in baboon models of gram negative sepsis (Taylor et al., 1987. J. Clin. Invest. 79; U.S. Pat. No. 5,009,889 to Taylor and Esmon) and preliminary clinical results suggest that protein C is effective in treating certain forms of human septic shock (Gerson et al., 1993. Pediatrics 91, 418-422). Inhibition of protein S, an important component of the protein C pathway, exacerbates the response of primates to sublethal levels of E. coli and augments the appearance of TNF in the circulation (Taylor et al., 1991. Blood 78, 357-363). These results suggest that protein C may both control coagulation and influence inflammation.
Protein C is activated when thrombin, the terminal enzyme of the coagulation system, binds to an endothelial cell surface protein, thrombomodulin (Esmon, 1989. J. Biol. Chem. 264, 4743-4746; Dittman and Majerus, 1990. Blood 75, 329-336; Dittman, 1991. Trends Cardiovasc. Med. 1, 331-336). In cell culture, thrombomodulin transcription is blocked by exposure of endothelial cells to tumor necrosis factor (TNF) (Conway and Rosenberg, 1988. Mol.Cell. Biol. 8, 5588-5592) and thrombomodulin activity and antigen are subsequently internalized and degraded (Lentz et al., 1991. Blood 77, 543-550, Moore, et.al., 1989. Blood 73, 159-165). C4bBP, a regulatory protein of the complement system, binds protein S to form a complex that is functionally inactive in supporting APC anticoagulant activity in vitro (Dahlback, 1986. J. Biol. Chem. 261, 12022-12027) and in vivo (Taylor,et al., 1991). C4bBP behaves as an acute phase reactant (Dahlback, 1991. Thromb. Haemostas. 66, 49-61). Thus, proteins of this pathway not only appear to regulate inflammation, but they also interact with components that regulate inflammation, and they themselves are subject to down regulation by inflammatory mediators.
Endothelial cells play a critical role in the protein C pathway in that they express two of the known receptors responsible for efficient protein C activation, thrombomodulin and the endothelial protein C/APC receptor (EPCR) (Fukudome and Esmon. 1994. J. Biol. Chem. 269:26486-26491; Stearns-Kurosawa, et al. 1996. Proc. Natl. Acad. Sci. (USA) 93:10212-10216). Thrombomodulin (CD141) is a transmembrane cofactor that binds circulating thrombin with high affinity and the resultant enzyme-cofactor complex is required for physiologically relevant protein C activation rates (Esmon and Owen. 1981. Proc. Natl. Acad. Sci. (USA) 78:2249-2252; Dittman, W. A. 1991. Trends Cardiovasc. Med. 1:331-336).
EPCR is a recently identified receptor with significant homology to the CD1/MHC class 1 family (Fukudome and Esmon, 1994; Fukudome, et al. 1996. J. Biol. Chem. 271:17491-17498; Regan, et al. 1996. J. Biol. Chem. 271:17499-17503). The cloning and biological role of the endothelial cell receptor for protein C was described in PCT/US95/09636 by Oklahoma Medical Research Foundation, entitled "Cloning and Regulation of an Endothelial Cell Protein C/Activated Protein C Receptor". The protein was predicted to consist of 238 amino acids, which includes a 15 amino acid signal sequence at the N-terminus, and a 23 amino acid transmembrane region which characterizes the receptor as a type 1 transmembrane protein.
EPCR binds both protein C and APC with similar affinity (Kd.sub.app .about.30 nM) (Fukudome, et al., 1996) in the presence of calcium and facilitates protein C activation by presenting the protein C substrate to the thrombin-thrombomodulin activation complex on cell surfaces (Stearns-Kurosawa, et al., 1996). Both endothelial cell receptors are type 1 transmembrane proteins in which the ligand binds to an extracellular domain and both have a short intracellular cytoplasmic tail (Fukudome, et al. 1996; Jackman, et al. 1987. Proc. Natl. Acad. Sci. (USA) 84:6425-6429; Wen, et al., 1987. Biochemistry 26:4350-4357; Suzuki, et al. 1987. EMBO J. 6:1891-1897). In addition, their in vitro cell surface expression is down-regulated similarly by tumor necrosis factor-.alpha.(Fukudome and Esmon 1994). However, the characteristics of soluble forms of thrombomodulin and EPCR differ in several respects. Recombinant soluble thrombomodulin has reduced cofactor activity relative to the membrane form (Galvin, et al. 1987. J. Biol. Chem. 262:2199-2205; Parkinson, et al. 1990. J. Biol. Chem. 265:12602-12610). With both purified components and with cells, the changes in thrombin's substrate specifically induced by thrombomodulin result from competition for a shared binding domain on thrombin as well as conformational alterations in the active site pocket (Ye, et al. 1991. J. Biol. Chem. 266:23016-23021; Lu, et al. 1989. J. Biol. Chem. 264:12956-12962; Ye, et al. 1992. J. Biol. Chem. 267:11023-11028; Hofsteenge, et al. 1986. Biochem. J. 237:243-251; Mathews, 1994. Biochemistry 33:13547-13552; Esmon, et al. 1982. J. Biol. Chem. 257:7944-7947; Sadler, et al. 1993. Haemostasis 23:183-193). Soluble thrombomodulin also accelerates inactivation of thrombin by a variety of inhibitors (Bourin and Lindahl. 1993. Biochem. J. 289:313-330; Rezaie, 1995. J. Biol. Chem. 270:25336-25339). Both plasma and urine contain detectable thrombomodulin (Takano, et al. 1990. Blood. 76:2024-2029; Ishii and Majerus. 1985. J. Clin. Invest. 76:2178-2181) and because the thrombomodulin gene does not contain introns (Jackman, et al., 1987), these soluble forms are due to proteolysis of the extracellular domain at the cell surface.
Soluble degradation products of thrombomodulin in plasma are a known marker of endothelial cell damage in a variety of disease states (Takano, et al., 1990; Tanaka, et al. 1991. Clin. Chem. 37:269-272; Takahashi, et al. 1991. Am. J. Hematol. 38:174-177; Asakura, et al. 1991. Am. J. Hematol. 38:281-287; Wada, et al. 1992. Am. J. Hematol. 39:20-24; Takahashi, et al. 1992. Am. J. Hematol. 41:32-39; Ohdama, et al. 1994. Chest 106:666-671) and are comprised of a mixture of thrombin-binding fragments with varying reduced affinities, as well as non-binding fragments (Takano, et al., 1990).
In contrast, recombinant soluble EPCR (rsEPCR), truncated just before the transmembrane domain, binds both protein C and APC with an affinity similar to that observed for intact cell-surface expressed EPCR (Fukudome, et al. 1996). APC anticoagulant activity is inhibited effectively when bound to rsEPCR (Regan, et al., 1996), presumably because both rsEPCR and factor Va share binding determinants in a groove reminiscent of the anion binding exosite I in thrombin occupied by thrombomodulin (Mather, et al. 1996. EMBO J. 15:6822-6831). However, rsEPCR does not appear to influence proteolysis of small synthetic substrates by APC, nor inactivation of APC by .alpha.1-antitrypsin or protein C inhibitor (Regan, et al., 1996). Unlike membrane-bound EPCR which enhances protein C activation (Stearns-Kurosaw, at al., 1996), rsEPCR has little effect on protein C activation by the soluble thrombin-thrombomodulin complex (Regan, et al., 1996), suggesting that any soluble forms of EPCR might inhibit protein C activation by competing with membrane-associated EPCR for protein C.
Immunohistochemistry indicates that EPCR is present primarily on the surface of endothelial cells from large vessels and is absent or present at low levels on most capillary endothelial cells.
It is therefore an object of the present invention to identify therapeutic and diagnostic uses for naturally occurring soluble EPCR.
It is a further object of the present invention to characterize naturally occurring soluble EPCR.